Field of the Invention
Certain aspects of the present invention generally relate to devices and methods for the electrochemical detection and quantitation of analyte in a sample.
Background
Point of care (POC) diagnostic tests are convenient and inexpensive devices useful for the detection of a target molecule, in a sample. POC tests are employed in a variety of settings, including for medical, consumer, or environmental purposes because of the low cost, ease of use, and, portability that they afford. POC tests have become increasingly popular in part because of the rapidity with which results are achieved and for the flexibility of configuring the devices for a variety of applications.
The lateral flow assay (LFA) is a POC diagnostic tool that is capable of determining the presence or absence of an analyte, typically, through visual observation of a discernible color change. One common example of such LFA tests is the conventional household pregnancy test. LFAs generally involve the use of a labeled antibody deposited at a first position on a solid substrate. Sample is applied to the first position, causing the labeled antibody to reconstitute in solution, whereupon the antibody binds to complementary analyte in the sample. Alternatively, labeled antibody is mixed with the sample prior to application on the LFA. In either case, a complex of analyte and antibody forms. The analyte-antibody complex flows along the liquid front from the first location through the solid substrate to a second location, a test line, where an immobilized antibody is located. The immobilized antibody captures the analyte through specific interaction, resulting in a high concentration of labeled antibody at the test line. The high concentration of labeled antibody provides a detectable visual signal. Gold nanoparticles are typically used to label the antibodies because they are relatively inexpensive and provide easily observable color indications based on the surface, plasmon resonance properties of gold nanoparticles. In many cases, this signal is used to, provide only, qualitative, information, such as whether or not the analyte is present in the sample.
FIGS. 1A and 1B depict a conventional one-step sandwich LFA 12, as described in the prior art. The sandwich LFA 12 is comprised of a substrate 13 over or through which the liquid sample flows. The sandwich LFA 12 comprises an upstream region 15 and a downstream region 17. Deposited on the substrate 13 at a first, region 14 are antibodies 24 conjugated to a visually or optically detectable particle 25, such as a gold nanoparticle. When a sample is applied to the upstream region 15 of the substrate 13, it may, flow over or through the substrate 13 to the downstream region 17 of the substrate 13. The labeled antibodies are reconstituted by the sample liquid when the sample flows to and through the first region 14. The labeled antibodies are reactive to a first epitope on an analyte of interest 27 which may be present in the sample, such that the labeled antibodies bind the analyte 27 when the sample passes through the first region 14.
The substrate 13 also comprises a second region 16, where capture antibodies 26 are deposited. The capture antibodies 26 are solubilized by the sample when the sample flows to and through the second region 16. The capture antibodies have biotin 29 conjugated thereto, and are reactive to a second, different epitope on the antigen 27, such that the capture antibodies 26 also bind to the analyte 27 when the analyte 27 passes through the second region 16. The analyte 27 is therefore bound by both the labeled antibodies 24 and the capture antibodies 26, forming a “sandwich” complex. The sandwich complex flows with the sample to a third region 18 forming a test line. The third region 18 has avidin 28 immobilized thereon. The avidin 28 binds to the biotin 29 on the capture antibodies 26, thereby retaining the sandwich complex at the third region 18, resulting in the accumulation of label particles 25 at the third region 18, which provides for a visual determination of the presence of analyte 27 in the sample. Although this sandwich detection method has been widely used, it is also possible to immobilize the capture antibodies 26 at the third region 18 for trapping labeled antibody-antigen complexes at the third region 18, and eliminating the second region 16.
Labeled antibodies 24 that are not bound to analyte 27 continue to flow past the third region 18 to an optional fourth region 20 forming a control line. The fourth region 20 has immobilized antibodies deposited thereon which bind to the labeled antibodies (e.g. species specific antibodies). Thus, when the analyte of interest 27 is present in the sample, a visual signal may be detected both at the third region 18 and at the fourth region 20. However, if the analyte 27 is not present in the sample, a visual signal is detected only at the fourth region 20.
FIG. 1B is a schematic representation of the binding interactions that take place at the third region 18. Labeled antibodies bind to a first epitope on the analyte 27 in the sample. Capture antibodies 26 with biotin 29 conjugated thereon binds to a second epitope on the analyte 27, forming the sandwich complex. Avidin 28 is immobilized on the third region 18, and bind to the biotin that is conjugated to the capture antibodies 26. The result is the capture of the sandwich complex, thereby providing the visual signal indicating the presence of the analyte 27 in the sample.
Recent efforts in the field of LFAs have focused on creating devices capable of quantitating the amount of analyte present in the sample. Such information is important, for example, when the amount of analyte in sample, rather than merely its presence, provides the required information. For example, a baseline level of analyte may be normal, but elevated analyte may be indicative of disease when it exceeds some threshold level.